Metabonomics approach to determine metabolic differences between green tea and black tea consumption

The purpose of this study was to compare the effects of black and green tea consumption on human metabolism. Seventeen healthy male volunteers consumed black tea, green tea, or caffeine in a randomized crossover study. Twenty-four-hour urine and blood plasma samples were analyzed by NMR-based metabonomics, that is, high-resolution 1H NMR metabolic profiling combined with multivariate statistics. Green and black tea consumption resulted in similar increases in urinary excretion of hippuric acid and 1,3-dihydroxyphenyl-2-O-sulfate, both of which are end products of tea flavonoid degradation by colonic bacteria. Several unidentified aromatic metabolites were detected in urine specifically after green tea intake. Interestingly, green and black tea intake also had a different impact on endogenous metabolites in urine and plasma. Green tea intake caused a stronger increase in urinary excretion of several citric acid cycle intermediates, which suggests an effect of green tea flavanols on human oxidative energy metabolism and/or biosynthetic pathways.     

A new process to develop a cocoa powder with higher flavonoid monomer content and enhanced bioavailability in healthy humans

Cocoa is a food rich in polyphenols, mainly the flavonoid procyanidins and flavan-3-ols. The improvement of the cardiovascular function in humans upon cocoa consumption has been specifically linked to the presence of flavan-3-ol derived metabolites in plasma, especially epicatechin glucuronide. In this context, a flavonoid-enriched cocoa-derived product could potentially exert stronger health benefits. The aim of the present study was to obtain a cocoa powder with a higher flavonoid content (mainly enriched in monomer compounds) and assess its flavonoid bioavailability in humans. For this purpose, an unfermented, nonroasted, and blanch-treated cocoa powder (A) was obtained. The powder contained four times more procyanidins than a conventional (B) cocoa powder. Powder A contained eight times more epicatechin and procyanidin B2 than powder B. Cocoa milk drinks were prepared with powder A (MDA) and B (MDB). The bioavailability of flavonoids in both drinks was assessed in a crossover intervention with healthy volunteers. The content of epicatechin glucuronide, the main metabolite detected in plasma, was five-fold higher upon consumption of MDA as compared with MDB. The urinary excretion of metabolites, mainly methyl epicatechin sulfate, was higher upon MDA consumption as compared with MDB, ranging from two- to 12-fold higher depending on the metabolite. These results, together with previous reports regarding the cardiovascular benefits linked to the presence of procyanidin metabolites in plasma, suggest that further clinical trials to validate the health benefits of a flavonoid-enriched cocoa powder are warranted.     

Metabolism of grape seed polyphenol in the rat

The metabolism of grape seed polyphenol (GSP) has been investigated in rats by high-performance liquid chromatography analysis of the serum and urinary concentrations of the GSP metabolites (+)-catechin (CT), (-)-epicatechin (EC), 3'-O-methyl-(+)-catechin, and 3'-O-methyl-(-)-epicatechin. The serum concentration of these four metabolites reached a maximum 3 h after the oral administration of GSP. The urinary excretion of these GSP metabolites accounted for 0.254% (w/w) of the administered dose of GSP (1.0 g/kg), and the majority of these metabolites were excreted within 25 h of oral administration. The serum concentration and urinary excretion of these metabolites were also compared after the oral administration of different GSP monomers (gallic acid, CT, and EC), normal GSP, and the high molecular weight components of GSP (GSPH). No metabolites were detected in the serum of rats given GSPH. The urinary percentage excretion of the GSP metabolites derived from the respective monomers (CT or EC) did not vary with the administration of different substances (CT or EC, GSP, or GSPH). Taken together, these results suggest that only the monomers of GSP are absorbed and metabolized.     

Nuclear magnetic resonance spectroscopic based studies of the metabolism of black tea polyphenols in humans

Epidemiological studies indicate that a high intake of flavonoids is associated with an improved health status. Tea is one of the most abundant sources of flavonoids in the human diet. The bioavailability and biotransformation of tea flavonoids are, however, not clearly understood. The aim of the present study was to investigate the metabolism of black tea via a nonspecific screening method. (1)H nuclear magnetic resonance (NMR) spectroscopy was used to obtain nonselective profiles of urine samples collected from three human volunteers before and after a single dose of black tea. The complex spectroscopic profiles were interpreted with the use of pattern recognition techniques. Hippuric acid was confirmed as the major urinary black tea metabolite. One previously unknown metabolite was detected and identified as 1,3-dihydroxyphenyl-2-O-sulfate (sulfate conjugate of pyrogallol) using HPLC directly coupled to mass spectrometry and (1)H NMR spectroscopy. This study shows that NMR-pattern recognition studies can be used for the discovery of unknown flavonoid metabolites in humans.     

Effect of simultaneous consumption of milk and coffee on chlorogenic acids' bioavailability in humans

Different studies have shown that milk may interact with polyphenols and affect their bioavailability in humans. The present study investigated the effect of the simultaneous consumption of coffee and milk on the urinary excretion of chlorogenic acids (CGA) and metabolites. Subjects were submitted to consumption of water, instant coffee (609 mmol of CGA) dissolved in water, and instant coffee dissolved in whole milk. Urine was collected for 24 h after consumption of each treatment for analysis of CGA and metabolites by HPLC/LC-MS. The amount of CGA and metabolites recovered after consumption of combined coffee-milk (40% ± 27%) was consistently lower in all subjects compared to that of coffee alone (68% ± 20%). Concluding, the simultaneous consumption of milk and coffee may impair the bioavailability of coffee CGA in humans.     

Effect of dietary nitrogen content on the urine metabolite profile of dairy cows assessed by nuclear magnetic resonance (NMR)-based metabolomics

NMR-based metabolomics was applied on urine samples from 32 cows that were fed four levels of crude protein (124, 135, 151, and 166 g/kg DM, respectively) in a crossover design with the aim of identifying urinary metabolites related to nitrogen intake and nitrogen efficiency. Principal component analysis (PCA) on selected regions of the obtained (1)H NMR spectra revealed an effect of crude protein intake on NMR signals in the 0.5-3.0 and 5.0-10.0 ppm regions. Partial least-squares (PLS) regressions confirmed a correlation between the NMR metabolite profile and both nitrogen intake and efficiency. The NMR signals that correlated with nitrogen intake and efficiency included urea, hippurate, phenylacetylglutamine, and p-cresol sulfate, which all contributed to the prediction of nitrogen intake and efficiency. Thus, it was not possible to identify a single metabolite that could be used as a marker to predict nitrogen efficiency, and it can be concluded that a wide-ranging urinary metabolite profile is needed to evaluate nitrogen efficiency in ruminants.     

1H NMR based metabolic profiling in the evaluation of Japanese green tea quality

Classification of tea quality is now mainly performed according to the sensory results by professional tea tasters. However, this evaluation method is inconsistent in differentiating their qualities. A combination of a (1)H NMR technique and a multivariate analysis was introduced to the quality evaluation of green tea by means of a metabolomic technique. A broad range of metabolites were detected by (1)H NMR spectrometry. The principal component analysis (PCA) was used to reduce the complexity of the (1)H NMR spectra data set and provided the quality discrimination result. It offered an extensive clue for classification and quality assessment without any prepurification method. A set of green teas from a Japanese tea contest were analyzed by (1)H NMR to classify the quality with respect to that judged by tea tasters and to conceive a quality prediction model. Metabolic profiling and fingerprinting of (1)H NMR spectra of green teas with different quality were studied. PCA showed a separation between the high- and the low-quality green teas. The taste marker compounds contributing to the discrimination of tea quality were identified. Reliable prediction models were obtained by the partial least-squares projection to latent structure (PLS) analysis together with a preprocessing filter of both orthogonal signal correction (OSC) and a combination between OSC and wavelet transform algorithms.     

Puerins A and B, two new 8-C substituted flavan-3-ols from Pu-er tea

Pu-er tea is a special treated fermented tea produced from crude green tea, which is prepared from the leaves of Camellia sinensis var. assamica. It is a traditional beverage having been used in China, particularly the southern areas, for a long time. Chemical investigation led to the identification of two new 8-C substituted flavan-3-ols, puerins A (1) and B (2), and two known cinchonain-type phenols, epicatechin-[7,8-bc]-4alpha-(4-hydroxyphenyl)-dihydro-2(3H)-pyranone (3) and cinchonain Ib (4), together with other seven known phenolic compounds, 2,2',6,6'-tetrahydroxydiphenyl (5), (-)-epicatechin-8-C-beta-d-glucopyranoside (6), (-)-epicatechin (EC) (7), (-)-epigallocatechin (EGC) (8), (+/-)-gallocatechin (GC) (9), gallic acid (10), and myricetin (11), in addition to caffeine (12). Their structures were determined by spectroscopic methods. The compounds 1-5, which might be formed in the postfermentative process of Pu-er tea, were isolated from tea and theaceous plants for the first time.     

Metabolic dependence of green tea on plucking positions revisited: a metabolomic study

The dependence of global green tea metabolome on plucking positions was investigated through (1)H nuclear magnetic resonance (NMR) analysis coupled with multivariate statistical data set. Pattern recognition methods, such as principal component analysis (PCA) and orthogonal projection on latent structure-discriminant analysis (OPLS-DA), were employed for a finding metabolic discrimination among fresh green tea leaves plucked at different positions from young to old leaves. In addition to clear metabolic discrimination among green tea leaves, elevations in theanine, caffeine, and gallic acid levels but reductions in catechins, such as epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG), glucose, and sucrose levels were observed, as the green tea plant grows up. On the other hand, the younger the green tea leaf is, the more theanine, caffeine, and gallic acid but the lesser catechins accumlated in the green tea leaf, revealing a reverse assocation between theanine and catechins levels due to incorporaton of theanine into catechins with growing up green tea plant. Moreover, as compared to the tea leaf, the observation of marked high levels of theanine and low levels of catechins in green tea stems exhibited a distinct tea plant metabolism between the tea leaf and the stem. This metabolomic approach highlights taking insight to global metabolic dependence of green tea leaf on plucking position, thereby providing distinct information on green tea production with specific tea quality.     

Pharmacokinetics and urine metabolite identification of dehydroevodiamine in the rat

This study investigates the oral bioavailability and characterizes urine metabolites of dehydroevodiamine (DeHE), one of the bioactive alkaloids isolated from the fruit of Evodia rutaecarpa . A freely moving rat model coupled with an automated blood sample system was used to evaluate the pharmacokinetics of DeHE. High-performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectrometry were applied to determine DeHE and its metabolites. The averaged oral bioavailability of DeHE (100 and 500 mg/kg) in the freely moving rats was approximately 15.35%. Cumulative fecal and urinary excretions of unchanged DeHE were 6 and 0.5%, respectively, after a single oral dose (500 mg/kg) of DeHE. The protein binding of DeHE in rat plasma was 65.6 ± 6.5%. Six metabolites, including five DeHE-O-glucuronides and one DeHE-sulfate, were identified after oral administration. The structures of two glucuronide conjugates, DeHE-10-O-glucuronide (M3) and DeHE-11-O-glucuronide (M4), and one sulfate conjugate, DeHE-12-sulfate (M6), were assigned. The findings indicate that the oral bioavailability of DeHE was much higher than that of evodiamine, and hydroxylation and conjugative metabolism were essential for the urinary elimination of DeHE.     

Age-related variations in flavonoid intake and sources in the Australian population

OBJECTIVE: To estimate flavonoid intake in the Australian population. DESIGN: Flavonoid consumption was estimated from 24-hour recall data and apparent consumption data using US Department of Agriculture flavonoid composition data. SUBJECTS: The National Nutrition Survey 1995 assessed dietary intake (24-hour recall) in a representative sample (n=13,858) of the Australian population aged 2 years and over. RESULTS: Analysis of the 24-hour recall data indicated an average adult intake (>18 years) of 454 mg day(-1) (92% being flavan-3-ols). Apple was the highest quercetin source until age 16-18 years, after which onion became an increasingly important prominent source. Variations in hesperetin consumption reflected orange intake. Apple, apricot and grapes were the major sources of epicatechin and catechin for children, but subsided as wine consumption increased in adulthood. Wine was the main source of malvidin. Naringenin intake remained static as a percentage of total flavonoid intake until age 19-24 years, corresponding to orange intake, and then increased with age from 19-24 years, corresponding to grapefruit intake. Apparent dietary flavonoid consumption was 351 mg person(-1) day(-1), of which 75% were flavan-3-ols. Black tea was the major flavonoid source (predominantly flavan-3-ols) representing 70% of total intake. Hesperetin and naringenin were the next most highly consumed flavonoids, reflecting orange intake. Both 24-hour recall and apparent consumption data indicated that apigenin intake was markedly higher in Australia than reported in either the USA or Denmark, presumably due to differences in consumption data for leaf and stalk vegetables and parsley. CONCLUSIONS: Tea was the major dietary flavonoid source in Australia. Flavonoid consumption profiles and flavonoid sources varied according to age. More consistent methodologies, survey tools validated for specific flavonoid intakes and enhanced local flavonoid content data for foods would facilitate better international comparisons of flavonoid intake.     

Urolithins Are the Main Urinary Microbial-Derived Phenolic Metabolites Discriminating a Moderate Consumption of Nuts in Free-Living Subjects with Diagnosed Metabolic Syndrome

Walnuts ( Juglans regia L.), hazelnuts ( Corylus avellana L.), and almonds ( Prunus dulcis Mill.) are rich sources of ellagitannins and proanthocyanidins. Gut microbiota plays a crucial role in modulating the bioavailability of these high molecular weight polyphenols. However, to date there are no studies evaluating the capacity to produce nut phenolic metabolites in subjects with metabolic syndrome (MetS), a pathology associated with an altered gut bacterial diversity. This study applied a LC-MS targeted approach to analyze the urinary excretion of nut phenolic metabolites in MetS subjects following 12 weeks of nut consumption, compared to sex- and age-matched individuals given a nut-free control diet. Metabolites were targeted in both hydrolyzed and nonhydrolyzed urine by LC-PDA-QqQ-MS/MS analysis, and identification of metabolites lacking available standards was confirmed by LC-ESI-ITD-FT-MS. Ellagitannin-derived urolithins A and B significantly increased after the nut-enriched-diet, urolithins C and D were also detected, and a complex combination of urolithin-conjugated forms was observed in nonhydrolyzed urine, confirming an extensive phase II metabolism after absorption. In contrast, no significant increases in proanthocyanidin microbial metabolites were observed in urine following nut consumption. Because the intestinal microbiota of the subjects in this study could catabolize ellagitannins into a wide range of urolithins, further research is strongly warranted on the in vivo potential of these microbial metabolites in reducing cardiometabolic risk.     

Metabolism of 7-fluoro-6-(3,4,5,6-tetrahydrophthalimido)-4- (2-propynyl)-2H-1,4-benzoxazin-3(4H)-one (S-53482, flumioxazin) in the rat: II. Identification of reduced metabolites

On single oral administration of (14)C-S-53482 [7-fluoro-6-(3,4,5, 6-tetrahydrophthalimido)-4-(2-propynyl)-2H-1,4-benzoxazin-3( 4H)-one, Flumioxazin] labeled at the 1- and 2-positions of tetrahydrophthaloyl group to rats at 1 (low dose) or 100 (high dose) mg/kg, the radiocarbon was almost completely eliminated within 7 days after administration in both groups with generally very low residual (14)C tissue levels. The predominant excretion route was via the feces. The major fecal and urinary metabolites involved reduction or sulfonic acid addition reactions at the 1,2-double bond of the 3,4,5,6-tetrahydrophthalimide moiety and hydroxylation of the cyclohexene or cyclohexane ring. One urinary and four fecal metabolites were identified using chromatographic techniques and spectroanalyses (NMR and MS). Three of five identified metabolites were unique forms, reduced at the 1,2-double bond of the 3,4,5, 6-tetrahydrophthalimide moiety. On the basis of the metabolites identified in this study, the metabolic pathways of S-53482 in rats are proposed. To specify tissues forming reduced metabolites, an in vitro study was conducted. Reduction was found to take place in red blood cells.     

Association between consumption of cruciferous vegetables and condiments and excretion in urine of isothiocyanate mercapturic acids

A high intake of cruciferous vegetables is associated with a reduced risk of cancer and cardiovascular diseases. This protective effect has been linked to isothiocyanates, enzymatic hydrolysis products of glucosinolates. In this study, the metabolic fate of glucosinolates and isothiocyanates after ingestion of 19 different cruciferous vegetables was studied in three male subjects. After the consumption of 13 cruciferous vegetables (glucosinolate content, 0.01-0.94 mmol/kg) and six condiments (isothiocyanate content, 0.06-49.3 mmol/kg), eight different isothiocyanate mercapturic acids were determined in urine samples. Excretion levels after the consumption of raw vegetables and condiments were higher (bioavailability, 8.2-113%) as compared to cooked vegetables (bioavailability, 1.8-43%), but the excretion rate was similar (t1/2=2.1-3.9 h). Isothiocyanates in urine remain longer at a nonzero level after the consumption of glucosinolates from cooked vegetables, as compared to raw vegetables and condiments, and maximal levels in urine were reached about 4 h later. Isothiocyanate mercapturic acids can be used as a biomarker to reflect the active dose of isothiocyanates absorbed.     

Metabolite profiling of Angelica gigas from different geographical origins using 1H NMR and UPLC-MS analyses

Angelica gigas obtained from different geographical regions was characterized using (1)H nuclear magnetic resonance (NMR) spectroscopy and ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) followed by multivariate data analyses. Principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) score plots from (1)H NMR and UPLC-MS data sets showed a clear distinction among A. gigas from three different regions in Korea. The major metabolites that contributed to the discrimination factor were primary metabolites including acetate, choline, citrate, 1,3-dimethylurate, fumarate, glucose, histamine, lactose, malate, N-acetylglutamate, succinate, and valine and secondary metabolites including decursin, decursinol, nodakenin, marmesin, 7-hydroxy-6-(2R-hydroxy-3-methylbut-3-ethyl)coumarin in A. gigas roots. The results demonstrate that (1)H NMR and UPLC-MS-based metabolic profiling coupled with chemometric analysis can be used to discriminate the geographical origins of various herbal medicines and to identify primary and secondary metabolites responsible for discrimination.     

A metabonomic strategy for the detection of the metabolic effects of chamomile (Matricaria recutita L.) ingestion

A metabonomic strategy, utilizing high-resolution 1H NMR spectroscopy in conjunction with chemometric methods (discriminant analysis with orthogonal signal correction), has been applied to the study of human biological responses to chamomile tea ingestion. Daily urine samples were collected from volunteers during a 6-week period incorporating a 2-week baseline period, 2 weeks of daily chamomile tea ingestion, and a 2-week post-treatment phase. Although strong intersubject variation in metabolite profiles was observed, clear differentiation between the samples obtained before and after chamomile ingestion was achieved on the basis of increased urinary excretion of hippurate and glycine with depleted creatinine concentration. Samples obtained up to 2 weeks after daily chamomile intake formed an isolated cluster in the discriminant analysis map, from which it was inferred that the metabolic effects of chamomile ingestion were prolonged during the 2-week postdosing period. This study highlights the potential for metabonomic technology in the assessment of nutritional interventions, despite the high degree of variation from genetic and environmental sources.     

A new LC/MS/MS rapid and sensitive method for the determination of green tea catechins and their metabolites in biological samples

A new rapid and sensitive method has been developed, using liquid chromatography in tandem mass spectrometry (LC-ESI-MS/MS) to identify green tea catechin metabolites in plasma and urine after oral intake of a green tea extract. (-)-Epigallocatechin-3-gallate (EGCG), (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin (EGC)-glucuronide, (-)-epicatechin (EC)-glucuronide, and EC-sulfate were identified in plasma, whereas in urine only the conjugated catechins were detected (EGC-glucuronide, EGC-sulfate, EC-glucuronide, and EC-sulfate). Standard calibration curves prepared in plasma were found to be linear in the range of 10.9-1379.3 nmol/L for EGCG, EGC, ECG, and EC. The accuracy and precision of this assay showed a coefficient of variation of <15%. The method allowed the detection and quantification limits (for 20 microL injection) from 1.1 to 2.6 nmol/L and 3.8-8.7 nmol/L, respectively, in plasma and 0.8-1.8 nmol/L and 2.6-6.0 nmol/L, respectively, in urine. This method can be applied for future clinical and epidemiological studies, allowing the identification of the active metabolites that will reach the target tissues.     

Absorption and urinary excretion of quercetin,rutin, and alphaG-rutin,a water soluble flavonoid,in rats

Quercetin, rutin, alphaG-rutin (a water soluble flavonoid), and a mixture of rutin and alphaG-rutin were administered to rats by a single gastric intubation, and their absorption and urinary excretion were examined. The plasma and 24 h urinary levels of aglycons (quercetin and tamarixetin/isorhamnetin) were measured by HPLC after deconjugation with beta-glucuronidase/sulfatase treatment. alphaG-rutin was absorbed more rapidly than quercetin or rutin, and the plasma concentrations of quercetin and tamarixetin/isorhamnetin reached the highest peak level 30 min after dosing. Quercetin, rutin, and the mixture of rutin and alphaG-rutin showed the first peak level 8 h, 8 h, and 30 min after dosing, respectively. The area under the concentration-time curve (AUC) for quercetin in rats administered alphaG-rutin was approximately 4.5- and 2-fold higher than those in rats administered quercetin and rutin, respectively, and was almost the same as that in rats administered a mixture of rutin and alphaG-rutin. The highest 24 h urinary excretion was observed in alphaG-rutin-administered rats. These results suggest that alphaG-rutin is absorbed more efficiently than either quercetin or rutin and that a high plasma concentration can be maintained by supplying rutin and alphaG-rutin in combination.     

High-throughput technique for comprehensive analysis of Japanese green tea quality assessment using ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/TOF MS)

Applications of metabolomics techniques along with chemometrics provide an understanding in the relationship between metabolome of green tea and its quality. A coupled of ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/TOF MS) allowed a high-throughput and comprehensive analysis with minimal sample preparation. Using this technique, a wide range of metabolites were investigated. Data analysis was rapid, considering that the fingerprinting technique was performed. A set of green tea samples from 2006 tea contest of the Kansai area was analyzed to prove usefulness of the developed technique. Green tea with different qualities were discriminated through principal component analysis (PCA). Consequently, projection to latent structure by means of partial least-squares (PLS) was performed to create a constructive quality-predictive model by means of metabolic fingerprinting. Beside epigallocatechin, other predominant catechins, including epigallocatechin gallate and epicatechin gallate, detected in green tea were found to be significant biomarkers to the high quality of Japanese green tea (Sencha).